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Fig. 6
Beta-cells differentiating from post-embryonic progenitors are more proliferative compared to the embryonic beta-cells. a Schematic showing the labeling of the embryonic beta-cell (EBC) population during growth. Tg(ins:Cre-ER T2 )—mediated recombination excises the floxed mCherry from Tg(ins:Red-Stop-Green). Beta-cells that undergo successful recombination activate insulin:H2B-GFP expression. Recombination was induced using a 4-OHT treatment at 3 dpf. New beta-cells that differentiate post-recombination are GFP-negative and can only express mCherry. The samples were stained at 30 dpf using an antibody for PCNA, which peaks in expression during the G1/S-stages of the cell cycle. b Confocal projections of islets from Tg(ins:Cre-ER T2 );Tg(ins:Red-Stop-Green) animals at 30 dpf. The GFP-positive beta-cells localize preferentially in the posterior half of the islet, whereas the anterior half is occupied by GFP-negative but mCherry-positive beta-cells. b′ Higher magnification single planes from the cells shown in b. A majority of the mCherry-positive but GFP-negative beta-cells are also PCNA-positive (yellow arrows) whereas only one of the GFP-positive beta-cells is PCNA-positive (white arrows). Note that since Tg(ins:Red-Stop-Green) contains multiple cassettes within one genomic integration site, some of the beta-cells are both GFP and mCherry-positive due to incomplete excision of mCherry in all cassettes during the 4-OHT treatment. c Quantification showing that a higher proportion of GFP-negative beta-cells were PCNA-positive at 30 dpf compared to the GFP-positive beta-cells, indicating higher proliferative capacity of de novo beta-cells during islet growth (n > 50 GFP-positive and -negative cells per islet, n = 7 islets) (unpaired two-tailed t-test, ***p < 0.005). Plot shows mean ± S.E.M. d Confocal projections of islets from Tg(ins:FUCCI-G1); Tg(ins:FUCCI-S/G2/M) animals at 23, 27, and 35 dpf oriented along the anterior–posterior axis. Samples were collected ~10 h after feeding, which is important to stimulate beta-cell proliferation during the late larval and juvenile stages in zebrafish33. e Quantification of the percentage of Tg(ins:FUCCI-S/G2/M)-positive and Tg(ins:FUCCI-G1)-negative beta-cells in the anterior and posterior halves of islets from each stage. At 23 and 27 dpf, a higher proportion of proliferating beta-cells are present in the islet’s anterior half, as compared to the posterior. At 35 dpf, beta-cell proliferation is similar in both the anterior and the posterior regions. In addition, beta-cell proliferation is reduced compared to 23 and 27 dpf (n = 8 islets at 23 dpf; n = 5 islets each at 27 and 35 dpf) (paired two-tailed t-test, *p < 0.05). Scale bars, 20 µm